Inflammatory bowel disease (IBD) alters neuronal regulation of the gastrointestinal (GI) tract. The superior mesenteric ganglia (SMG) contain sympathetic neurons that modulate GI functions such, as motility and blood flow. IBD reduces the release of noradrenaline, a sympathetic neurotransmitter. We hypothesized that the reduction in NA release is due to inhibition of voltage-gated calcium current (ICa), as calcium influx is a regulator of neurotransmitter release. We also hypothesized that tumor necrosis factor α (TNFα), a proinflammatory cytokine elevated during IBD, can also inhibit the ICa of SMG neurons. Therefore, we compared ICa amplitude in neurons from normal mice and mice with dextran sulphate sodium (DSS; 5% w/v)-induced colitis. Neurons dissociated from the SMG were cultured overnight and changes to ICa were investigated using electrophysiological, Ca2+ imaging, PCR and neurotransmitter release techniques. Colitis significantly reduced ICa of SMG neurons by selectively inhibiting N-type Ca2+ channels. This was accompanied by a reduction in mRNA encoding the N-type channel alpha subunit (CaV 2.2) and a rightward shift in the voltage dependence of activation of ICa. Colitis reduced the NA release from the colon and jejunum. Depolarization-induced release of tritiated-NA was inhibited by ω-Conotoxin GVIA (300 nM). These results suggest that the changes in VGCC observed at the cell bodies of SMG neurons were also occurring at the nerve terminals during colitis. Similar experimental techniques were performed using SMG neurons incubated overnight in TNFα (1nM). TNFα decreased ICa and depolarization-induced Ca2+ influx in SMG neurons. Similar to DSS-induced colitis, the reduction in ICa was limited to N-type Ca2+ channels. Preincubation of neurons with SC 514 (20μM) and Bay 11 7082 (1µM), inhibitors of nuclear factor kappa B signaling, prevented the reduction in ICa. Preincubation with the p38 MAPK inhibitor, PD 169316 (30µM), recovered a smaller portion of the reduction in Ca2+ influx. These data suggest that DSS colitis and TNFα inhibit N-type VGCC ICa in sympathetic neurons and identify a novel role for NF-κB and p38 MAPK in the regulation of neurotransmitter release. These findings also suggest that DSS colitis inhibits NA release by altering sympathetic N-type VGCC in the colon and jejunum. / Thesis (Master, Physiology) -- Queen's University, 2008-09-02 12:06:20.438

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The kinetics of production and role of tumor necrosis factor-alpha (TNF-$ alpha$) and nitric oxide (NO) during the early phase of blood-stage infection with Plasmodium chabaudi AS were investigated using two inbred strains of mice which differ in the level of resistance to this parasite. Analysis of the in vivo expression of TNF-$ alpha$ and inducible nitric oxide synthase (iNOS) revealed that, early during infection, resistant C57BL/6 mice, which clear the infection by 4 weeks, have higher levels of TNF-$ alpha$ and iNOS mRNA in the spleen and TNF-$ alpha$ mRNA in the liver than susceptible A/J mice which succumb to the disease 10 days after initiation of infection. Moreover, resistant mice expressed high levels of IFN-$ gamma$ (a Th1 marker) and low levels of IL-4 (a Th2 marker) mRNA in the spleen, whereas susceptible A/J mice had low levels of IFN-$ gamma$ but high levels of IL-4 mRNA in the spleen early during infection. Increased levels of NO$ sb3 sp-$ were detected in serum of resistant C57BL/6 mice only at the time of peak parasitemia. Furthermore, treatment of resistant C57BL/6 mice with anti-IFN-$ gamma$ and anti-TNF-$ alpha$ monoclonal antibody demonstrated that TNF-$ alpha$, either alone or in synergy with IFN-$ gamma$, plays a major role in the up-regulation of NO production during P. chabaudi AS malaria. Moreover, treatment with the iNOS inhibitor aminoguanidine, eliminated resistance of these mice to infection with P. chabaudi AS without affecting parasitemia, suggesting that NO may not be involved in parasite killing in vivo. Taken together, these results demonstrate that a Th1-associated increase in TNF-$ alpha$ early during infection, as occurs in resistant mice, leads to the up-regulation of NO production which is crucial for survival of the host. On the other hand, our results also suggest that a Th2 response, as occurs in susceptible mice, does not result in protective levels of TNF-$ alpha$ and NO. However, susceptible A/J mice were found to

Typescript (Photocopy) / Bibliography: leaves 202-281 / x, 281 leaves, 2 leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--Dept. of Medicine, University of Adelaide, 1990

[Formulae and special characters can only be approximated. Please see the pdf version of this abstract for an accurate reproduction.] Tumor necrosis factor (TNF) is a pleiotropic cytokine that mediates a long list of immunological and pathophysiological processes. TNF is produced by a wide variety of cells including immune and non-immune cells, however in most cell types TNF is not expressed prior to stimulation. The function of TNF is mediated via its trimeric domain by binding to TNF receptors that are found on most types of cells, especially of the haematopoietic systems, hence transpiring its effects on a wide variety of cells and organ systems. The cytotoxic (apoptosis) and pro-inflammatory (differentiation, proliferation and activation) functions of TNF are protective but can also result in pathological or deleterious consequences. A biallelic G to A transition polymorphism in the promoter region of TNF at nucleotide position 308 from the transcription start site is suggested to be involved in differential transcriptional regulation of TNF expression. The high TNF producing 308A allele is associated with susceptibility to or worse outcome of many infectious diseases in addition to autoimmune and other pathophysiological conditions. A previous study in our laboratory observed a selective affinity towards the polymorphic 308A allele by an EMSA protein(s) complex, named E. Several other protein complexes were found along with complex E and one of them was identified as Sp1. The identification of complex E was unsuccessful but it was hypothesized to play a major role as transcriptional activator in 308A allele individuals hence transpiring its effect in various pathophysiological states. In this study, the EMSA complexes observed in the TNF promoter region between nucleotides 322 to 283, encompassing the 308 polymorphism, is characterized. EMSA using mutated oligonucleotides mapped the binding sites of complexes B, C, D and E. TRANSFAC database search in addition to previous work revealed the identity of complex C as Sp1 but the rest of complexes remained unknown. Moreover, in contrast to our previous study, the protein(s) in the complex E was found to preferentially bind 308G nucleotide hence posing as a transcriptional repressor, resulting in decreased production state of TNF in 308G allele individuals than 308A allele individuals. In order to characterize putative transcription factors binding to the promoter region, first the biochemical characteristics such as the effects of temperature, salts and cations on DNA binding ability of EMSA complexes were studied. EMSA complexes B, C, DI and E required cations, probably Zn+2, to bind DNA. By optimizing a technique that couples EMSA with SDS-PAGE, the molecular weight of C, DI and E was determined. A novel technique that couples EMSA with IEF determined the pI of complexes B, C, D, DI and E. Although a commonly used technique of identifying unknown DNA-binding protein of interest, Yeast One-Hybrid assay, did not identify complex E, the novel identification method involving chromatography, two-dimensional electrophoresis, EMSA, mass spectrometry and database interrogation successfully identified TNF EMSA complex E as transcription factor Ying Yang 1 (YY1). Supershift EMSA confirmed complex E as YY1. In addition, the supershift assay showed presence of Sp1 and Sp3 in complex C. Similarly, complex DI is identified as Sp3. The novel method in identifying DNA-binding proteins is particularly useful as this technique allows identification of protein seen in EMSA without the need of extensive identification process. YY1 binds to a 6 base pair sequence, 5? TTGAGG 3?, from nt 295 to 290 of TNF promoter. The loss of affinity in 308A allele is caused by transition of underlined G nucleotide to A. The determined and described molecular weight of YY1 in literature is 60 kDa while the theoretical weight is 45 kDa. Both the determined and theoretical pI of YY1 is 5.8. YY1 is a multifunctional transcription factor implicated in both positive and negative regulation of gene expression as well as in initiation of transcription. It is ubiquitously expressed in growing, differentiated, and growth-arrested cells. Although future experiment is yet to establish in vivo presence of YY1 in TNF promoter, our study so far provides convincing evidence that the putative transcription factor that has selective affinity towards 308G allele is indeed YY1.

[Truncated abstract] Tumor Necrosis Factor (TNF) is a potent pro-inflammatory cytokine involved in a range of biological functions including the differentiation, proliferation and survival of many cell types. The TNF gene lies in the class III region of the major histocompatibility complex (MHC), approximately 250 Kbp centromeric of the HLA-B locus and 850 Kbp telomeric of HLA-DR. Due to the genomic location and biological relevance of TNF, it is thought that genetic heterogeneity at this locus may be associated with autoimmune and infectious diseases. A G-to-A single nucleotide polymorphism (SNP) at position -308 (relative to the transcriptional start site) in the TNF promoter has been well described. The less common -308A variant has been shown to be linked with the HLA-A1, B8, DR3 haplotype which in turn has been associated to a high TNF producing phenotype. Determining whether the -308 polymorphism contributes to elevated levels of expression has therefore been a priority for many research groups. Some investigators have shown differences in transcription between the -308G and -308A alleles while others could not. These contradicting results have led to conflicting views regarding the functional relevance of the -308 SNP. In this study, statistical analysis of 18 independent transient transfections of -308 biallelic TNF reporter constructs have provided evidence for a functional consequence of the polymorphism. ... In addition, chromatin accessibility of this region was maximal at greater levels of transcription suggesting a role for both chromatin structure and YY1 binding in -308G regulation. Surprisingly, chromatin structure did not seem to play a role in -308A regulation nor was there any significant binding of YY1, suggesting the -308 region does not affect transcriptional control of TNF. Taken as a whole, the G-to-A SNP relieves YY1 binding and demonstrates an allele-specific regulatory mechanism controlling expression. A growing list of promoter polymorphisms exists in the human genome having associations with certain diseases. Determining the functional consequence of these SNPs has proven difficult and utilized mainly in vitro approaches. In this thesis, a unique approach to investigating the functionality of promoter polymoprhisms has been developed, utilizing in vivo techniques which test their effects in a more natural system. It is hoped that the identification of the allele-specific YY1-mediated control of the -308 region of the TNF promoter may provide insight into overexpression as a consequence of the polymorphism and its role in the genetic susceptibility to MHC-associated autoimmune disease.

Thesis (M.S.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. "August 2007" Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 106-118).

Thesis (Ph.D. in Cancer Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 112-124). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;